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Transient α-helices in the disordered RPEL motifs of the serum response factor coactivator MKL1.

Mizuguchi M, Fuju T, Obita T, Ishikawa M, Tsuda M, Tabuchi A - Sci Rep (2014)

Bottom Line: Proline mutagenesis showed that the transient α-helices are locally formed without helix-helix interactions.The helix content is higher in the order of RPEL1, RPEL2, and RPEL3.The amount of preformed structure may correlate with the binding affinity between the intrinsically disordered protein and its target molecule.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Structural Biology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan.

ABSTRACT
The megakaryoblastic leukemia 1 (MKL1) protein functions as a transcriptional coactivator of the serum response factor. MKL1 has three RPEL motifs (RPEL1, RPEL2, and RPEL3) in its N-terminal region. MKL1 binds to monomeric G-actin through RPEL motifs, and the dissociation of MKL1 from G-actin promotes the translocation of MKL1 to the nucleus. Although structural data are available for RPEL motifs of MKL1 in complex with G-actin, the structural characteristics of RPEL motifs in the free state have been poorly defined. Here we characterized the structures of free RPEL motifs using NMR and CD spectroscopy. NMR and CD measurements showed that free RPEL motifs are largely unstructured in solution. However, NMR analysis identified transient α-helices in the regions where helices α1 and α2 are induced upon binding to G-actin. Proline mutagenesis showed that the transient α-helices are locally formed without helix-helix interactions. The helix content is higher in the order of RPEL1, RPEL2, and RPEL3. The amount of preformed structure may correlate with the binding affinity between the intrinsically disordered protein and its target molecule.

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ΔδCα - ΔδCβ secondary chemical shifts of (a) RPEL2 and (b) RPEL3.The positions of helices α1 and α2 are indicated in each panel.
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f6: ΔδCα - ΔδCβ secondary chemical shifts of (a) RPEL2 and (b) RPEL3.The positions of helices α1 and α2 are indicated in each panel.

Mentions: We also investigated the conformational propensity of RPEL2 and RPEL3 using NMR spectroscopy (Figure 6). The residues from Thr132 to Lys139 and from Leu149 to Met 152 of RPEL2 have a propensity to adopt an α-helical conformation (Figure 6a). On the other hand, RPEL3 exhibits no significant helical propensity (Figure 6b). Together, our results suggest that the helices α1 and α2 are transiently formed in RPEL1 and RPEL2, while the helix is not formed in RPEL3. The helix content is higher in the order of RPEL1 > RPEL2 > RPEL3 (Figures 4 and 6).


Transient α-helices in the disordered RPEL motifs of the serum response factor coactivator MKL1.

Mizuguchi M, Fuju T, Obita T, Ishikawa M, Tsuda M, Tabuchi A - Sci Rep (2014)

ΔδCα - ΔδCβ secondary chemical shifts of (a) RPEL2 and (b) RPEL3.The positions of helices α1 and α2 are indicated in each panel.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048911&req=5

f6: ΔδCα - ΔδCβ secondary chemical shifts of (a) RPEL2 and (b) RPEL3.The positions of helices α1 and α2 are indicated in each panel.
Mentions: We also investigated the conformational propensity of RPEL2 and RPEL3 using NMR spectroscopy (Figure 6). The residues from Thr132 to Lys139 and from Leu149 to Met 152 of RPEL2 have a propensity to adopt an α-helical conformation (Figure 6a). On the other hand, RPEL3 exhibits no significant helical propensity (Figure 6b). Together, our results suggest that the helices α1 and α2 are transiently formed in RPEL1 and RPEL2, while the helix is not formed in RPEL3. The helix content is higher in the order of RPEL1 > RPEL2 > RPEL3 (Figures 4 and 6).

Bottom Line: Proline mutagenesis showed that the transient α-helices are locally formed without helix-helix interactions.The helix content is higher in the order of RPEL1, RPEL2, and RPEL3.The amount of preformed structure may correlate with the binding affinity between the intrinsically disordered protein and its target molecule.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Structural Biology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan.

ABSTRACT
The megakaryoblastic leukemia 1 (MKL1) protein functions as a transcriptional coactivator of the serum response factor. MKL1 has three RPEL motifs (RPEL1, RPEL2, and RPEL3) in its N-terminal region. MKL1 binds to monomeric G-actin through RPEL motifs, and the dissociation of MKL1 from G-actin promotes the translocation of MKL1 to the nucleus. Although structural data are available for RPEL motifs of MKL1 in complex with G-actin, the structural characteristics of RPEL motifs in the free state have been poorly defined. Here we characterized the structures of free RPEL motifs using NMR and CD spectroscopy. NMR and CD measurements showed that free RPEL motifs are largely unstructured in solution. However, NMR analysis identified transient α-helices in the regions where helices α1 and α2 are induced upon binding to G-actin. Proline mutagenesis showed that the transient α-helices are locally formed without helix-helix interactions. The helix content is higher in the order of RPEL1, RPEL2, and RPEL3. The amount of preformed structure may correlate with the binding affinity between the intrinsically disordered protein and its target molecule.

Show MeSH
Related in: MedlinePlus